Helga Kolb - US grants

The primary goal of this research is to understand the neural pathways that form ganglion cell receptive fields in the mammalian and ultimately the human retina. Most of the techniques to be used in our investigations are anatomical but we continue an ongoing collaboration with Dr. Ralph Nelson (NINCDS) who uses physiological approaches to address the same questions. Major advances in our understanding of rod and cone pathways and ON and OFF center pathways in the mammalian retina have been gained by elucidating neural circuitry of physiologically identified, HRP-injected cells in cat retina, and we intend to continue with these same approaches. Electron microscopic studies of the synaptic input to X, Y and W type ganglion cells, of amacrine and bipolar cells is ongoing and will continue as more cells become available from the physiological experiments. To further our understanding of rod pathways in the mammalian retina we will continue a cross species analysis of Golgi impregnated neurons using opossum, ground squirre, rabbit, monkey and human retinas. Differences between mammalian retinas that have developed a visual streak rather than a fovea as a specialization will be investigated further by comparisons of Golgi impregnated rabbit and ground squirrel retinas to those of cat. Ganglion cells of these species will be marked with DAPI and Lucifer dye or HRP injection to show their morphology and orientation relative to the visual streak. Immunocytochemical studies on the cat retina, using antibodies to neurotransmitter candidates or their synthetic enzymes will be done in collaboration with Dr. Harvey Karten (Stony Brook). Thus cells that stain for TH, substance P, VIP, GAD and cholecystokinin will be stained in whole-mount, characterised at the light microscope level by comparison with Golgi impregnated counterparts and investigated by electron microscopy for synaptology. Human retina available from the Lions Eye Bank and from surgical procedures in the Ophthalmology Department will be studied by light (Golgi, fluorescence) and EM techniques (serial section analysis) to investigate the following a) morphology of the blue cones, b) distributions of rod amacrine cells and synaptology of the rod system and c) synaptology of the midget and parasol ganglion cells in comparison with cat beta ganglion cells.

It is proposed to study the synaptic connections of the neurons in the outer plexiform layer of the turtle retina by electron microscopy. Long series (100-500) of ultrathin sections will be examined, and profiles of interest identified in the inner nuclear layer and followed to their synaptic connections with identified photoreceptor cells. The different types of horizontal cells will be distinguished on cytological criteria and their synaptic relationships (chemical and electrical synapses) with other horizontal or bipolar cells will be evaluated. Amacrine-like profiles which have been seen in the outer plexiform layer will be followed in serial section to determine whether they originate from interplexiform cells. Golgi-impregnated bipolar cells that can be considered the morphological counterparts of physiologically identified on-center and off-center bipolar types will be sectioned and examined by electron microscopy for the morphology of their synaptic junctions. At the same time, it will be possible to study the contacts of the impregnated bipolar cells with the different spectral types of cones. Due to the poor preservation of Golgi-impregnated material, it will be necessary to examine these bipolar-photoreceptor contacts in greater detail by looking at serial sections of well-fixed, conventionally prepared material. Interactions between the different spectral types of cones will also be investigated by serial section electron microscopy of Golgi-impregnated and horseradish-peroxidase injected photoreceptors. The purpose of this study is to gain an understanding of the morphological basis for functional pathways originating at the outer plexiform layer. Furthermore, since spectral types of cones are readily identifiable in the turtle retina, valuable information concerning color coding at the first synaptic level will be attainable.

Our major long-term objective is to understand the synaptic connections of the neurons that underlie visual processing in the vertebrate retina and, to ascertain how the visual image is integrated into parallel ganglion cell pathways for transmission to the brain. Our studies over the years have used the turtle retina in addition to mammalian retinas mainly because certain functional pathways have often been more easily elucidated in this cold-blooded, highly visually-dependent vertebrate: particularly pathways concerning color. It turns out that morphological and physiological examination of turtle retina together with pharmacological and behavioral studies have been proceeding so rapidly in the last decade, that the turtle retina is now one of the best studied in retinal research. We have been major contributors to these studies and we think that we can continue to integrate anatomical and electrophysiological findings with greater advantage in this retina than in others. The emphasis of this grant proposal is directed at seeking general principles of retinal organization concerning tangentially and radially organized mosaics and pathways involved in spectral processing. Specifically we will: 1) Investigate whether the different spectral types of photoreceptor are arranged into repeating units or an organized mosaic; 2) Determine the spectral inputs and ultrastructure of synaptic contacts to cone pedicles of intracellularly recorded and HRP -injected color-coded bipolar cells and see how they fit into the photoreceptor mosaic; 3) Look at spectral contacts of chromaticity H2, H3 and H4 cells with the objective of linking the color-coded bipolar and horizontal cells into pathways arising from the photoreceptor mosaic; 4) Ascertain whether Muller cells divide patches of retina into continuous "columns" of organized color units from photoreceptors through bipolar cells to a group of ganglion cells subserving this column, and 5) In collaboration with Richard Normann and Josef Ammermuller, who are recording turtle ganglion cell responses with multiple electrode arrays, attempt to understand the architecture of color- coded ganglion cell that are involved in their recordings.

Our major long-term objective is to understand the synaptic connections of the neurons that underly complex visual functions such as color vision, directional selectivity and orientation specificity in the vertebrate retina. For this purpose the turtle retina is particularly advantageous because a) the spectral types of cone can be recognized on morphological criteria so that chromatic wiring to second order neurons can be readily studied, and 2) the retina with its linear visual streak organization is particularly specialized for motion and orientation detection. Moreover, the turtle retina has been a favorite experimental retina for electrophysiologists because stable long-term intracellular recordings are possible from all the retinal neurons with subsequent marking of the neurons with HRP for anatomical investigations. Using anatomical techniques of studying serial semi-thin and ultrathin sections for electron microscopy (EM) and reconstructing neurons and portions of the neuropil we intend to: 1) determine the arrangement and ultrastructural characteristics of the various spectral types of cone pedicle in the outer plexiform layer with particular emphasis on the blue and ultraviolet sensitive cone pedicles. 2) Subsequently we will determine the chromatic input to C-type horizontal cells and the unknown H4 type of the turtle retina and various bipolar cell types. 4) In addition we will determine the connections of the Golgi-- stained centrifugal bipolar in the OPL to see whether this cell might be an interplexiform cell. 5) Using immunocytochemical staining of bipolar cells we will study which chromatic type they might be and study their output to amacrine and ganglion cells in the inner plexifonil layer. Simultaneously we will continue examining HRP-marked and physiologically -identified 6) amacrine and 7) ganglion cells for synaptology of their inputs and outputs. We will concentrate particularly on color-coded and directionally-selective and motion-sensitive amacrine and ganglion cells. We will use EM immunocytochemistry in combination with serial section EM to determine what the neurotransmitter specific input and output neurons might be that are involved with these HRP-marked amacrine and ganglion cells. Finally we will determine the synaptic circuitry of substance P amacrine and ganglion cells as well as attempt to discover which ganglion cell types these might be, by double labeling of retrogradely rhodamine-bead labeled ganglion cells.